title: Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.
abstract: Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.
authors.affiliation: Mechanobiology Institute, National University of Singapore.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Fu
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Freedman
authors.forename: Chwee Teck
authors.initials: CT
authors.lastname: Lim
authors.forename: Rebecca
authors.initials: R
authors.lastname: Heald
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
mesh_terms: D000818:Animals; D002843:Chromatin / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D010063:Ovum / Q000648:ultrastructure; D014982:Xenopus laevis / Q000235:genetics*
chemical_list: D002843:Chromatin